Beginning in October, 1992, cases of cholera due to a new strain of Vibrio cholerae , cholerae O139 Bengal, were recognized in India; this strain has subsequently spread in epidemic form across Asia, portending the beginning of the eighth cholera pandemic. Persons with prior exposure to V. cholerae O1 are not protected against infection with O139 Bengal strains. V. cholerae O1 El Tor and V. cholerae O139 Bengal are extremely closely related phylogenetically, and carry the same virulence factors. However, O139 Bengal strains do not express the O1 antigen, and have acquired a polysaccharide capsule. Manning has shown that O139 Bengal strains have lost almost all of the O1 antigen biosynthetic complex (Vcrfb genes); we have identified a region of > 11 kb (designated cps) which does not appear to be present in O1 El Tor strains and is associated with encapsulation and expression of the "semi-rough" O139 Bengal O antigen side chain. Our primary objective is to understand the genetic changes which led to the emergence of V. cholerae O139 Bengal phenotype, and, in turn, permitted the strain to avoid immunologic detection by persons with prior immunity to O1 El Tor strains. We hypothesize that the critical genetic elements in this shift were the acquisition of the cps region, combined with deletion of Vcrfb. To provide a basis for subsequent genetic analysis, we will complete our structural characterization of the O1 39 Bengal capsular polysaccharide. We will define the sugar composition of the O antigen side chain to allow further exploration of the relationship between O antigen and capsule biosynthesis. At a genetic level, we will isolate and characterize the cps region, including sequencing, identification of open reading frames, and assessment of the impact of deletions within the region on expression of the capsule and the O antigen. We will then see if we can "recreate" the O139 phenotype by introduction of the cps region and deletion of Vcrfb from V, cholerae O1 El Tor. If we are unable to do so, it may be due to the presence of additional insertions/deletions outside of the cps and Vcrfb regions; we will use representational difference analysis and other techniques to look for such changes. Finally, by using a rabbit model, we will evaluate the effect of phenotypic changes associated with manipulation of cps and Vcrfb genes on development of protective immunity to V. cholerae O1, seeking to define the critical change(s) which have allowed V. cholerae O139 Bengal to spread in pandemic form through populations with prior immunity to V. cholerae 01.